Cell migration is a fundamental process required for embryogenesis, angiogenesis, wound healing and immune responses, and abnormalities in this process are associated with a plethora of pathological conditions including autoimmune diseases, arthrosclerosis and tumor metastasis. Cell migration is regulated by chemoattractants acting on cell-surface receptors belonging to the large family of G protein-coupled receptors. These receptors typically couple to G proteins of the Gi/o class, and mediate their functions through Gbg dimers. Although the essential role of Gbg in cell migration has been well established, it is still unclear how Gbg activate downstream effectors to generate a highly polarized intracellular signal that acts as an internal compass to drive directional cell migration. We have recently identified novel interactions between Gbg and the WD40 repeat proteins RACK1 (receptor for activated C kinase 1) and WDR26. Based on the findings that RACK1 and WDR26 are localized at the leading edge of a polarized leukocyte and negatively and positively regulate Gbg signaling, respectively, we hypothesize that reciprocal interactions of Gbg with WDR26 and RACK1 are important positive and negative feedback regulations for the coordinated Gbg signaling at the leading edge that drives leukocyte polarization and directed migration. In Aim 1, we will explore the molecular mechanism by which WDR26 interacts with Gbg and its effectors including PI3Ks and PLCb to promote Gbg signaling and generate a highly polarized intracellular response at the leading edge for leukocyte migration. In Aim 2, we will determine how RACK1 restricts Gbg signaling for the generation of a locally amplified signal at the leading edge and for signal adaptation and directional sensing. Finally, we will dissect the molecular signals that control the reciprocal interactions of WDR26 and RACK1 with Gbg and determine the impact of the balance of these interactions on leukocyte migration. These studies have the potential to close a critical gap in our understanding of how chemoattractants transmit through Gbg to generate a spatially localized cellular signal at the leading edge that drives leukocyte migration, and to unveil a novel mechanism of regulating chemotaxis at the level of G proteins upstream to all known signaling pathways essential for cell migration. Given the involvement of abnormal leukocyte migration in many pathological conditions, the proposed research will be vital for identifying novel targets to enable selective interference of the signaling pathways related to disease development. Thus, this work has high biological significance and potential impact on human health.